BioFiles Volume 5, Number 2 — Protein Characterization & Detection

Download Biofiles v5 n2 (3.4 Mb PDF)


Table of Contents


X-ray Crystallography/Crystallization



Investigating protein function and interaction, as well as developing direct drug design strategies requires structural information provided by X-ray crystallography. The Nobel Prize in Chemistry 2009 was awarded to V. Ramakrishnana, T. Steitz and A. Yonath for their work in the field of structural and functional studies of ribosomes. X-ray crystallography was the applied method most often used during these studies. The recent award emphasizes the importance of this and similar techniques.

The elucidation of a macromolecular structure at the atomic level by X-ray or neutron diffraction analysis requires the compound to be formed into relatively large single crystals without any inclusions. Protein crystallization is very difficult because of the fragile nature of protein crystals. Attempting to crystallize a protein without a proven protocol can be very tedious. Many parameters that influence the crystallization process are not completely understood and require particular consideration. The crystallization process is still empirical and based mainly on trial end error.

In brief, precipitation occurs when a dissolved solid compound (solute) separates from its containing solvent. Precipitated solids may be structurally amorphous and heterogeneous, with weak molecular structures. These precipitants may have occluded solvent or include coprecipitated impurities also present in the solution. Crystallization is a specific precipitation process that occurs when the molecules of the solute grow slowly in well-organized forms that develop over time, reducing the likelihood of impurities being incorporated into the structure. The solute molecules are arranged in a uniform three dimensional structure, resulting in a crystal with greater rigidity and purity than an amorphous precipitate.

The process of crystallization can be differentiated into two steps:

  1. Nucleation process
  2. Crystal growth

where the nucleation process needs a higher degree of supersaturation than is needed for crystal growth.

Many soluble proteins, membrane proteins, nucleic acids, and nucleoprotein complexes have been obtained in a crystalline form suitable for crystallographic investigation.1–12 When a solution of a biopolymer is brought to supersaturation, the biopolymer may form crystals suitable for X-ray diffraction analysis, an amorphous precipitate, or any physical form between these two extremes. Parameters such as pH, temperature, chemical composition of the crystallization solution, and the rate of supersaturation determine whether an amorphous precipitate or crystals are formed.

Supersaturation is often achieved by increasing the concentration of precipitating agents in the crystallization solution. Auxiliary substances may improve crystallization or may initiate crystallization in otherwise static solutions. Larger structures, such as the purple membrane13 or ribosomal particles,14 can be grown into ordered two-dimensional structures in vitro. These structures can then be investigated by optical or electron diffraction and subsequent threedimensional image reconstruction with 7 Å resolution.

Precipitation Reagents

Any material which affects on the solubility of proteins can be used as a precipitant. Salts, organic solvents and polymers are typically used as precipitants to induce and optimize protein crystallization.

A limited number of salts can be used to produce protein and nucleic acid crystals. For most crystallizations it is essential to find the optimal salt concentration, which may be between 15 and 85% saturation. This value must be defined to a precision of 1–2%. Ammonium sulfate is the most widely used salt precipitant. Due to their chelating properties, citrate salts are especially useful when the presence of divalent cations interferes with crystallization of biopolymers. Cetyltrimethylammonium salts are often used with proteins and nucleic acids since macromolecule solubility in a crystallization solution increases with increasing salt concentration.

Organic Solvents
As with salt precipitants, organic solvent concentration must be calculated with a precision of 1–2% in order to crystallize a given biopolymer. Care must be used in selecting organic solvents for crystallization since, in contrast to salt precipitants, organic solvents can easily denature biopolymers. 2-Methyl-2,4-pentanediol, also known as MPD or hexylene glycol, is the most widely used organic solvent precipitant, as it has been found to be mild and efficient when used to crystallize sensitive macromolecules.

Certain polymers may produce an increase of the thermodynamic activity of a biopolymer-containing solution followed by phase separation. The most widely used polymers for crystallization are the polyethylene glycols (PEG). Proteins with higher molecular weights tend to precipitate from solution at lower concentrations of PEG.

Auxillary Substances
Additional substances for crystallization may be needed in certain situations. The presence of small polyamines such as spermine, cadaverine, spermidine or putrescine in the crystallization solution seems to aid in the growth of high quality transfer RNA crystals. These polyamines may act as specific counterions for the negatively charged phosphate groups. X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA15 have clearly shown the potential of this method.16

Except for the precipitant concentration, the most important variable in crystallization conditions is the pH value, as different pH values may result in a change of structural orientation. Buffers are often necessary for the maintenance of a specific pH. A large selection of buffers recommended for crystallography are available.

Since crystal growth generally requires time to reach completion, exposure to oxygen is likely to occur. In such cases, it is recommended to include a mild antioxidant such as cysteine, β-mercaptoethanol, glutathione, or dithiothreitol in the crystallization solution.

Metal ions may improve the crystallization of proteins irrespective of whether the ion is the cofactor of the apoprotein or not. Chelating agents, such as EDTA, should be added to the crystallization solution if metal cations prevent crystal formation. Furthermore, a variety of small molecules and ions have been found to affect the crystallization process.1

Crystallization Kits for Protein Analysis

Crystallography kits reduce the time and effort needed for the screening process. These kits are designed and qualified to help researchers determine the optimal conditions for growing protein crystals reliably and reproducibly. Using efficient sparse matrix screening, Sigma® crystallography kits are compatible with key crystallography techniques including:

  • Hanging, sitting, or sandwiched drops in vapor diffusion.
  • Free-layer or membrane-bound diffusion.

Each kit offers a combination of high-purity buffers, salts, and precipitants to allow the exploration of salting-out, salting-in, cryoresistance, or micelle-mediated crystallization of proteins. Optimized protocols are included to simplify and refine the initial screening and growing of macromolecular crystals. As a result, researchers can achieve reliable results with a minimal investment of time, effort, and macromolecular sample quantity.

Sigma's crystallization kits offer:

  • Variety of proven precipitants — Allow rapid and simplified screening across a broad range.
  • High-purity reagents — Ensure reliable, reproducible results.
  • 0.2 µm filtered solutions — Avoids waste of valuable samples.
  • Conveniently packaged combinations of reagents and solutions — Simplified scale-up of crystallization following determination of optimal conditions.

Optimization Reagents

Additional Kits

Labware for Crystallization


  1. McPherson, A., Preparation and Analysis of Protein Crystals, John Wiley and Sons, New York (1982).
  2. Section II: Crystallization and Treatment of Crystals, Methods Enzymol., 114, 49-196 (1985).
  3. Theory of protein solubility. Arakawa, T., and Timasheff , S.N., Methods Enzymol., 114, 49 (1985).
  4. Nucleation and growth of protein crystals: general principles and assays Feher, G. and Kam, Z., Methods Enzymol., 114, 77 (1985).
  5. Crystallization of macromolecules: general principles. McPherson, A., Methods Enzymol., 114, 112 (1985).
  6. Crystallization of ps. R. Giegi, in: Crystallography in Molecular Biology (D. Moras et al., eds.), p. 15, Plenum Press, New York (1987).
  7. Protein crystallization: the growth of large-scale single crystals. Gilliland, G.L. and Davies, D.R., Methods Enzymol., 104, 370 (1984).
  8. Determination of a transfer RNA structure by crystallographic method. Kim, S.-H. and Quigley, G.J., Methods Enzymol., 59, 3 (1979).
  9. Experimental neutron protein crystallography. Schoenborn, B.P., Methods Enzymol., 114, 510 (1985).
  10. The application of neutron crystallography to the study of dynamic and hydration properties of proteins. Kossiakoff , A.A., Ann. Rev. Biochem., 54, 1195 (1985).
  11. New directions in protein crystal growth. DeLucas, L.J. and Bugg, C.E., Trends Biotechnol., 5, 188 (1987).
  12. The future of protein crystal growth. Bugg, C.E., J. Cryst. Growth, 76, 535 (1986).
  13. Three-dimensional model of purple membrane obtained by electron microscopy. Henderson, R. and Unwin, P.N., Nature, 257, 28 (1975).
  14. The growth of ordered two-dimensional sheets of ribosomal particles from salt-alcohol mixtures. Arad, T, et al., Anal. Biochem., 167, 113 (1987).
  15. X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA. Kim, S.H., et al., J. Mol. Biol., 75, 421 (1973).
  16. High-resolution x-ray diff raction studies on a pure species of transfer RNA. Ladner, J.E., et al., J. Mol. Biol., 72, 99 (1972).

back to BioFiles v5 n2
back to top